Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, ...loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca2+- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca2+ retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca2+ and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca2+- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.
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•Mitochondrial SPG7 is essential for the PTP complex formation in multiple cell types•SPG7 interacts with CypD and VDAC at the IMM and OMM contact site•C terminus of SPG7 and CsA-binding region of CypD are necessary for PTP formation•Loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis
Using an RNAi-based screen, Shanmughapriya et al. identify mitochondrial spastic paraplegia 7 as essential for the PTP complex formation. They show that SPG7 interacts with CypD and VDAC1 at the IMM and OMM contact site, and loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis.
Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. ...Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.
Abstract
While treatments exist for the acute phase of stroke, there are limited options for patients with chronic infarcts and long-term disability. Allogenic mesenchymal stem cells (alloMSCs) show ...promise for the treatment of stroke soon after ischemic injury. There is, however, no information on the use of autologous MSCs (autoMSCs), delivered intracerebrally in rats with a chronic infarct. In this study, rats underwent middle cerebral artery occlusion (MCAO) to induce stroke followed by bone marrow aspiration and MSC expansion in a closed bioreactor. Four weeks later, brain MRI was obtained and autoMSCs (1 × 10
6
, 2.5 × 10
6
or 5 × 10
6
;
n
= 6 each) were stereotactically injected into the peri-infarct and compared to controls (MCAO only; MCAO + PBS;
n
= 6–9). Behavior was assessed using the modified neurological severity score (mNSS). For comparison, an additional cohort of MCAO rats were implanted with 2.5 × 10
6
alloMSCs generated from a healthy rat. All doses of autoMSCs produced significant improvement (54–70%) in sensorimotor function 60 days later. In contrast, alloMSCs improved only 31.7%, similar to that in PBS controls 30%. Quantum dot–labeled auto/alloMSCs were found exclusively at the implantation site throughout the post-transplantation period with no tumor formation on MRI or Ki67 staining of engrafted MSCs. Small differences in stroke volume and no differences in corpus callosum width were observed after MSC treatment. Stroke-induced glial reactivity in the peri-infarct was long-lasting and unabated by auto/alloMSC transplantation. These studies suggest that intracerebral transplantation of autoMSCs as compared to alloMSCs may be a promising treatment in chronic stroke.
We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-l-phenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction ...coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-l-phenylalanine (pCNPhe) and 4-azido-l-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photostability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe.
Two novel 2'-deoxyadenosine (dA) analogues, Si
-dA-SCN and Si
-dA-SeCN, and two novel phenylalanine (Phe) analogues, Boc-Me-PheCH
SCN and Boc-Me-PheCH
SeCN, have been synthesized and the thiocyanate ...(SCN) and selenocyanate (SeCN) functional groups evaluated as vibrational reporters. The syntheses of Si
-dA-SCN and Si
-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, and the syntheses of Boc-Me-PheCH
SCN and Boc-Me-PheCH
SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively. The SCN and SeCN stretch vibrational modes were shown to be sensitive to the local environment by frequency shifts and full-width half-maximum (fwhm) changes in response to tetrahydrofuran (THF) and THF/water solvent mixtures. The vibrational lifetimes of the Si
-dA-SeCN (237±12 ps) and Boc-Me-PheCH
SeCN (295±31 ps) in THF solution were determined by ultrafast infrared pump-probe spectroscopy to be 1.5 to 3 times longer than those for Si
-dA-SCN (140±6 ps) and Boc-Me-PheCH
SCN (102±4 ps). The longer lifetimes for the SeCN analogues were attributed to the better insulating effects of the heavier selenium atom compared to the sulfur atom. The solvent sensitivity and longer vibrational lifetimes compared to other vibrational reporters suggest that SCN and SeCN vibrational reporters are well suited to studying several dynamic processes including protein and nucleic acid hydration and conformational changes, however stability issues may require post-synthetic modification methods to incorporate these reporters into biomacromolecules.
Two novel 2′-deoxyadenosine (dA) analogues, Si
2
-dA-SCN and Si
2
-dA-SeCN, and two novel phenylalanine (Phe) analogues, Boc-Me-PheCH
2
SCN and Boc-Me-PheCH
2
SeCN, have been synthesized and the ...thiocyanate (SCN) and selenocyanate (SeCN) functional groups evaluated as vibrational reporters. The syntheses of Si
2
-dA-SCN and Si
2
-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, and the syntheses of Boc-Me-PheCH
2
SCN and Boc-Me-PheCH
2
SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively. The SCN and SeCN stretch vibrational modes were shown to be sensitive to the local environment by frequency shifts and full-width half-maximum (fwhm) changes in response to tetrahydrofuran (THF) and THF/water solvent mixtures. The vibrational lifetimes of the Si
2
-dA-SeCN (237 ± 12 ps) and Boc-Me-PheCH
2
SeCN (295 ± 31 ps) in THF solution were determined by ultrafast infrared pump-probe spectroscopy to be 1.5 to 3 times longer than those for Si
2
-dA-SCN (140 ± 6 ps) and Boc-Me-PheCH
2
SCN (102 ± 4 ps). The longer lifetimes for the SeCN analogues were attributed to the better insulating effects of the heavier selenium atom compared to the sulfur atom. The solvent sensitivity and longer vibrational lifetimes compared to other vibrational reporters suggest that SCN and SeCN vibrational reporters are well suited to studying several dynamic processes including protein and nucleic acid hydration and conformational changes, however stability issues may require post-synthetic modification methods to incorporate these reporters into biomacromolecules.
Infrared spectroscopy demonstrates that thiocyanate and selenocyanate groups are sensitive reporters with vibrational lifetimes in the 100-300 ps range.
Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary ...human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.
A number of institutions have clinically implemented CYP2D6 genotyping to guide drug prescribing. We compared implementation strategies of early adopters of CYP2D6 testing, barriers faced by both ...early adopters and institutions in the process of implementing CYP2D6 testing, and approaches taken to overcome these barriers.
We surveyed eight early adopters of CYP2D6 genotyping and eight institutions in the process of adoption. Data were collected on testing approaches, return of results procedures, applications of genotype results, challenges faced, and lessons learned.
Among early adopters, CYP2D6 testing was most commonly ordered to assist with opioid and antidepressant prescribing. Key differences among programs included test ordering and genotyping approaches, result reporting, and clinical decision support. However, all sites tested for copy-number variation and nine common variants, and reported results in the medical record. Most sites provided automatic consultation and had designated personnel to assist with genotype-informed therapy recommendations. Primary challenges were related to stakeholder support, CYP2D6 gene complexity, phenotype assignment, and sustainability.
There are specific challenges unique to CYP2D6 testing given the complexity of the gene and its relevance to multiple medications. Consensus lessons learned may guide those interested in pursuing similar clinical pharmacogenetic programs.
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